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We evaluated the specificity of the primers OPF-01, P54, and 1253 to identify A. fumigatus, A. flavus, and A. niger, respectively, with the RAPD-PCR method. Eighty-two isolates belonging to the sections Fumigati, Flavi, and Nigri were used. The isolates were identified by phenotypic (macro- and micromorphology) and genotypic (partial sequences of the BenA gene) methods. The RAPD-PCR method was used to obtain polymorphic patterns with the primers OPF-01, P54, and 1253. The specificity of the polymorphic patterns of the isolates of each species was evaluated through the UPGMA clustering method and logistic regression model. All isolates of the genus Aspergillus were identified at the section level by macro- and micromorphology showing the typical morphology of the sections Fumigati, Flavi, and Nigri, and the species were identified by the construction of the phylogeny of the partial sequence of the BenA gene. The patterns' polymorphic strains obtained with the primers OPF-01, P54, and 1253 for the isolates of A. fumigatus, A. flavus, and A niger, respectively, showed the same polymorphic pattern as the reference strains for each species. To verify the specificity of the primers, they were tested with other species from the sections Fumigati, Flavi and Nigri. The results support that the primers OPF-01, P54, and 1253 generate polymorphic patterns by RAPD-PCR species specific to A. fumigatus, A. flavus, and A. niger, respectively.

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In this study, molecular typing using Randomly Amplified Polymorphic DNA (RAPD-PCR) was conducted on 16 original isolates of Metarhizium acridum obtained from locusts (Schistocerca piceifrons ssp. piceifrons.) in Mexico (MX). The analysis included reference strains of the genus Metarhizium sourced from various geographical regions. The isolates were identified by phenotypic (macro and micromorphology) and genotypic methods (RAPD-PCR and Amplified Fragment Length Polymorphisms (AFLP), through a multidimensional analysis of principal coordinates (PCoA) and a minimum spanning network (MST). Subsequently, Sequences-Characterized Amplified Region (SCAR) markers were developed for the molecular detection of M. acridum, these markers were chosen from polymorphic patterns obtained with 14 primers via RAPD-PCR. Phenotypic and genotypic characterization identified the MX isolates as M. acridum. Of all the polymorphic patterns obtained, only OPA04 and OPA05 were chosen, which presented species-specific bands for M. acridum, and further utilized to create SCAR markers through cloning and sequencing of the specific bands. The specificity of these two markers was confirmed via Southern hybridization. The SCAR markers (Ma-160OPA-05 and Ma-151OPA-04) exhibit remarkable sensitivity, detecting down to less than 0.1 ng, as well as high specificity, as evidenced by their inability to cross-amplify or generate amplification with DNAs from other strains of Metarhizium (as Metarhizium anisopliae) or different genera of entomopathogenic fungi (Cordyceps fumosorosea and Akanthomyces lecanii). These SCAR markers yield readily detectable results, showcasing high reproducibility. They serve as a valuable tool, especially in field applications.

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Coccidioidomycosis, caused by Coccidioides immitis and C. posadasii, causes significant morbidity and mortality, both in immunocompetent and immunocompromised people, mainly in endemic areas. The present work analyzed its epidemiology, diagnostic methods, and treatment by reviewing clinical cases published from 1950 to 2021. Fifty-nine articles were included, corresponding to 275 clinical cases. The results showed a higher incidence of coccidioidomycosis in the male gender than the female gender. The most affected age group was 31-40 years, and the most reported clinical presentation was disseminated with greater involvement in cutaneous and subcutaneous tissue, followed by the CNS, bone system, and peritoneum. The species most frequently reported was C. immitis. The most used treatment was azoles, followed by their combination with amphotericin B, monotherapy with amphotericin B, and alternative medicine. This work shows that epidemiological data outside the USA are still scarce. Serological tests are the preferred diagnostic method in daily medical practice, and cultures remain the gold standard. The treatment for coccidioidomycosis is ketoconazole and amphotericin B, individually or in combination.

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Dermatophytes are fungi included in the genera Trichophyton, Microsporum, Epidermophyton, Nannizzia, Paraphyton, Lophophyton, and Arthroderma. Molecular techniques have contributed to faster and more precise identification, allowing significant advances in phylogenetic studies. This work aimed to identify clinical isolates of dermatophytes through phenotypic (macro- and micromorphology and conidia size) and genotypic methods (sequences of ITS regions, genes of ß tubulin (BT2), and elongation factor α (Tef-1α)) and determine the phylogenetic relationships between isolates. Ninety-four dermatophyte isolates from Costa Rica, Guatemala, Honduras, Mexico, and the Dominican Republic were studied. The isolates presented macro- and micromorphology and conidia size described for the genera Trichophyton, Microsporum, and Epidermophyton. Genotypic analysis classified the isolates into the genera Trichophyton (63.8%), Nannizzia (25.5%), Arthroderma (9.6%), and Epidermophyton (1.1%). The most frequent species were T. rubrum (26 isolates, 27.6%), T. interdigitale (26 isolates, 27.6%), and N. incurvata (11 isolates, 11.7%), N. gypsea and A. otae (nine isolates, 9.6%), among others. The genotypic methods clarified the taxonomic status of closely related species. For instance, the ITS and BT2 markers of T. rubrum/T. violaceum did not differ but the Tef-1α gene did. On the other hand, the three markers differed in T. equinum/T. tonsurans. Therefore, the ITS, BT2, and Tef-1α genes are useful for typing in phylogenetic analyses of dermatophytes, with Tef-1α being the most informative locus. It should be noted that isolate MM-474 was identified as T. tonsurans when using ITS and Tef-1α, but when using BT2, it was identified as T. rubrum. On the other hand, no significant difference was found when comparing the methods for constructing phylogenies, as the topologies were similar.

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COVID-19-associated pulmonary aspergillosis (CAPA) has had a high incidence. In addition, it has been associated with prolonged hospital stays, as well as several predisposing risk factors, such as fungal factors (nosocomial organism, the size of the conidia, and the ability of the Aspergillus spp. of colonizing the respiratory tract), environmental factors (remodeling in hospitals, use of air conditioning and negative pressure in intensive care units), comorbidities, and immunosuppressive therapies. In addition to these factors, SARS-CoV-2 per se is associated with significant dysfunction of the patient's immune system, involving both innate and acquired immunity, with reduced CD4+ and CD8+ T cell counts and cytokine storm. Therefore, this review aims to identify the factors influencing the fungus so that coinfection with SARS-CoV-2 can occur. In addition, we analyze the predisposing factors in the fungus, host, and the immune response alteration due to the pathogenicity of SARS-CoV-2 that causes the development of CAPA.

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As a result of the COVID-19 pandemic, various joint efforts have been made to support the creation of vaccines. Different projects have been under development, of which some are in the clinical evaluation stage and others in are in phase III with positive results. The aim of this paper was to describe the current situation of the development and production of vaccines available to the population to facilitate future research and continue developing and proposing ideas for the benefit of the population. So, we carried out a systematic review using databases such as PubMed, ScienceDirect, SciELO, and MEDLINE, including keywords such as "vaccines," "COVID-19," and "SARS-CoV-2". We reviewed the development and production of the anti-COVID vaccine and its different platforms, the background leading to the massive development of these substances, and the most basic immune aspects for a better understanding of their physiological activity and the immune response in those who receive the vaccine. We also analyzed immunization effects in populations with any medical or physiological conditions (such as immunosuppression, people with comorbidities, and pregnancy), as well as the response to immunization with heterologous vaccines and the hybrid immunity (the combination of natural immunity to SARS-CoV-2 with immunity generated by the vaccine). Likewise, we address the current situation in Mexico and its role in managing the vaccination process against SARS-CoV-2 at the national and international levels. There are still many clinical and molecular aspects to be described, such as the duration of active immunity and the development of immunological memory, to mention some of the most important ones. However, due to the short time since the global vaccination roll-out and that it has been progressive (not counting children and people with medical conditions), it is premature to say whether a second vaccination schedule will be necessary for the near future. Thus, it is essential to continue with health measures.

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The objective of this work was to use the random amplification of the polymorphic DNA-polymerase chain reaction (RAPD-PCR) technique to select polymorphic patterns through qualitative and quantitative analyses to differentiate the species A. flavus, A. fumigatus, A. niger and A. tubingensis. Twenty-seven Aspergillus isolates from different species were typified using phenotypic (macro- and micromorphology) and genotypic (partial BenA gene sequencing) methods. Thirty-four primers were used to obtain polymorphic patterns, and with these a qualitative analysis was performed to select the primers that presented species-specific patterns to distinguish each species. For the quantitative selection, a database was built from the polymorphic patterns and used for the construction of logistic regression models; later, the model that presented the highest value of sensitivity against specificity was evaluated through ROC curves. The qualitative selection showed that the primers OPA-19, P54, 1253 and OPA-02 could differentiate the species. A quantitative analysis was carried out through logistic regression, whereby a species-specific correlation of sensitivity and specificity greater than 90% was obtained for the primers: OPC-06 with a 96.32% match to A. flavus; OPF-01 with a 100% match to A. fumigatus; OPG-13 with a 98.01% match to A. tubingensis; and OPF-07 with a 99.71% match to A. niger. The primer OPF-01 discriminated the four species as well as closely related species. The quantitative methods using the selected primers allowed discrimination between species and showed their usefulness for genotyping some of the species of medical relevance belonging to the genus Aspergillus.

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Systemic candidiasis is a frequent opportunistic mycosis that can be life-threatening. Its main etiological agent is Candida albicans; however, the isolation of non-albicans Candida species has been increasing. Some of these species exhibit greater resistance to antifungals, so the rapid and specific identification of yeasts is crucial for a timely diagnosis and optimal treatment of patients. Multiple molecular assays have been developed, based mainly on polymerase chain reaction (PCR), showing high specificity and sensitivity to detect and identify Candida spp. Nevertheless, its application in diagnosis has been limited due to specialized infrastructure or methodological complexity. The objective of this study was to develop a PCR assay that detects and identifies some of the most common pathogenic Candida species and evaluate their diagnostic utility in blood samples and bronchial lavage. A pair of oligonucleotides was designed, CandF and CandR, based on sequence analysis of the 18S-ITS1-5.8S-ITS2-28S region of the rDNA of Candida spp., deposited in GenBank. The designed oligonucleotides identified C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, C. krusei/Pichia kudriazevii, C. guilliermondii/Meyerozyma guilliermondii, C. lusitaniae/Clavispora lusitaniae, and C. dubliniensis using simplex PCR based on the amplicon size, showing a detection limit of 10 pg/µL of DNA or 103 yeasts/mL. Based on cultures as the gold standard, it was determined that the sensitivity (73.9%), specificity (96.3%), and the positive (94.4%) and negative (81.2%) predictive values of the PCR assay with the designed oligonucleotides justify their reliable use in diagnosis.

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BACKGROUND: The molecular reclassification of the order Trichosporonales placed the medically relevant Trichosporon species into three genera of the family Trichosporonaceae: Cutaneotrichosporon, Trichosporon, and Apiotrichum. From the clinical and epidemiological standpoint, it is important to identify any species of the family Trichosporonaceae because they present different antifungal susceptibility profiles. In Mexico, little is known about trichosporonosis etiology because the fungi are identified through phenotypic methods. AIMS: To identify at a molecular level 12 yeast isolates morfologically compatible with Trichosporon, obtained from patients with superficial infections. METHODS: The yeast isolates were obtained from patients with white piedra, onychomycosis, and hand and foot dermatomycosis, and were identified morphologically and genotypically (sequencing of the IGS1 region and phylogenetic analysis using the Maximum Likelihood Method). The phylogenetic analysis included 40 yeast sequences from the order Trichosporonales and one from Cryptococcus neoformans as outgroup. RESULTS: Based on the molecular analysis, we identified three (25%) Trichosporon inkin isolates, two (16.7%) Trichosporon asteroides, two (16.7%) Cutaneotrichosporon mucoides, and one each (8.3%) of Trichosporon aquatile, Trichosporon asahii, Apiotrichum montevideense, Cutaneotrichosporon cutaneum, and Cutaneotrichosporon jirovecii. CONCLUSIONS: The molecular characterization of the isolates showed a broad diversity of species within the order Trichosporonales, particularly among onychomycosis. It is essential to identify these yeasts at the species level to delve into their epidemiology.

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BACKGROUND: The progressive disseminated histoplasmosis (PDH) has been associated with severe disease and high risk of death among people living with HIV (PLWHIV). Therefore, the purpose of this multicenter, prospective, double-blinded study done in ten Mexican hospitals was to determine the diagnostic accuracy of detecting Histoplasma capsulatum antigen in urine using the IMMY ALPHA Histoplasma EIA kit (IAHE), clarus Histoplasma GM Enzyme Immunoassay (cHGEI IMMY) and MiraVista Histoplasma Urine Antigen LFA (MVHUALFA); as well as the Hcp100 and 1281-1283220SCAR nested PCRs in blood, bone-marrow, tissue biopsies and urine. METHODOLOGY/PRINCIPAL FINDINGS: We included 415 PLWHIV older than 18 years of age with suspicion of PDH. Using as diagnostic standard recovery of H. capsulatum in blood, bone marrow or tissue cultures, or histopathological exam compatible, detected 108 patients (26%, [95%CI, 21.78-30.22]) with proven-PDH. We analyzed 391 urine samples by the IAHE, cHGEI IMMY and MVHUALFA; the sensitivity/specificity values obtained were 67.3% (95% CI, 57.4-76.2) / 96.2% (95% CI, 93.2-98.0) for IAHE, 91.3% (95% CI, 84.2-96.0) / 90.9% (95% CI, 87.0-94.0) for cHGEI IMMY and 90.4% (95% CI, 83.0-95.3) / 92.3% (95% CI, 88.6-95.1) for MVHUALFA. The Hcp100 nested PCR was performed on 393, 343, 75 and 297, blood, bone marrow, tissue and urine samples respectively; the sensitivity/specificity values obtained were 62.9% (95%CI, 53.3-72.5)/ 89.5% (95%CI, 86.0-93.0), 65.9% (95%CI, 56.0-75.8)/ 89.0% (95%CI, 85.2-92.9), 62.1% (95%CI, 44.4-79.7)/ 82.6% (95%CI, 71.7-93.6) and 34.9% (95%CI, 24.8-46.2)/ 67.3% (95%CI, 60.6-73.5) respectively; and 1281-1283220SCAR nested PCR was performed on 392, 344, 75 and 291, respectively; the sensitivity/specificity values obtained were 65.3% (95% CI, 55.9-74.7)/ 58.8% (95%CI, 53.2-64.5), 70.8% (95%CI, 61.3-80.2)/ 52.9% (95%CI, 46.8-59.1), 71.4% (95%CI, 54.7-88.2)/ 40.4% (95%CI, 26.4-54.5) and 18.1% (95%CI, 10.5-28.1)/ 90.4% (95%CI, 85.5-94.0), respectively. CONCLUSIONS/SIGNIFICANCE: The cHGEI IMMY and MVHUALFA tests showed excellent performance for the diagnosis of PDH in PLWHIV. The integration of these tests in clinical laboratories will certainly impact on early diagnosis and treatment.

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